It is well known that enzymes in liquid media can be stabilized using compounds such as metal halide salts (U.S. Pat. No. 7,157,416), alcohol ethoxylates (U.S. Pat. No. 4,548,727), aliphatic glycols and 1,3 propanediol (U.S. Pat. No. 3,819,528). The aim of these stabilization techniques is to stabilize substantially against loss of activity during storage. The ability to stabilize enzymes has been useful for users of industrial enzymes. Onsite storage in large vessels can reduce shipping costs, decrease the space required to store containers of enzyme and reduce the risk of enzyme shortages. Stabilization also reduces the risk of bacterial infections caused by microorganisms in the enzyme solution. For enzyme users that employ bioreactors and reagents with biological activity, such as producers of fuel ethanol, these infections can reduce product and co-product yield, impair process efficiency and increase operating costs incurred to fight the infections.
Currently, common industrial enzymes such as proteases, group 3 hydrolases and lipases are mixed with both metal halide salts such as sodium chloride and polyols, such as glycerol, before shipping to customers. Other compounds used for stabilization include antioxidants and amino acids such as methionine, which prevents oxidation of surface amino acids.
The following prior art provides examples of effective techniques for enzyme stabilization that have afforded substantial benefits to users of industrial enzymes. For example, Vermousek et al. (CS 210467) shows a powder mixture of urease with buffering, complexing and antibacterial agents that has practically infinite stability. Kelemen et al. (U.S. published patent application No. 20080152638) describe a glucose oxidase enzyme with improved storage stability. Becker et al. (U.S. Pat. No. 7,157,416) describe enzyme-containing formulations, comprising a metal halide salt and a polyol, having improved stability and enzymatic activity in liquid medium, particularly protease enzymes. Jaber (WO/2005117948) describes a method of preparing a stabilized bulk solution of a monomeric protein which consists in providing a bulk of monomeric protein in a buffer solution and adding an excipient to the bulk where the excipient is selected from the group of bacteriostatic agents, surfactants, isotonicity agents, amino acids, antioxidants and combinations thereof. Jaber's method prefers IFN-beta as the monomeric protein.
It has become commonplace for users of industrial enzymes to add the entire cocktail of enzyme and polyol and metal halide salt stabilizers and other preservatives into a bioreactor. There are a number of reasons for this, including the expense associated with separating enzymes from their metal halide salt, polymeric stabilizers and the requirement to separate enzyme from salt, polymeric stabilizers immediately prior to addition to a bioreactor so as to minimize instability and bacterial growth. In addition, very little has been published about the effects of dissociating these enzymes from their stabilizers prior to use in a bioreactor.
Bioreactors are now well known. In general, a bioreactor is a vessel in which a biochemical reaction takes place. Commercial-scale bioreactors typically have a capacity of over 1000 gallons. In commercial scale ethanol plants, bioreactors in which starch and cellulose are hydrolysed with enzymes typically have a capacity of 20,000 to 100,000 gallons. Fermentation vessels, within which enzymes catalyze biochemical reactions and microorganisms use reaction intermediates to produce metabolites, typically have a capacity of 100,000 to 1,000,000 gallons. Conditions such as temperature, pressure, pH and solution viscosity are tightly controlled within bioreactors due to the sensitivity of biochemicals and microorganisms. For example bioreactors within which starch and cellulose are hydrolysed typically have temperatures in the range of 75 to 100 degrees Celsius for starch and 45 to 75 degrees Celsius for cellulose.
One of the problems with adding the entire commercial enzyme formulation to the bioreactor is that inorganic salts will often contribute to instability of certain enzymes at high temperatures. A study by Klibanov showed that inorganic salts such as KCl and Na2SO4 destabilize thermostable alpha-amylase at 90 degrees Celsius. In addition, ions of inorganic salts can pose problems downstream from the bioreactor in final products. This is a serious problem in ethanol used for transportation fuel. For example, a study by Galante-Fox et al. shows that chloride levels greater than 3.5 ppm (weight) in fuel ethanol can cause severe corrosion of steel, reducing the demand for these transportation fuel products and other grain and cellulose-derived products.
There is a large body of knowledge concerning the use of enzymes in organic solvents and the benefits of pretreating these enzymes prior to use in organic solvents. Klibanov has shown that pretreating lipases prior to use in organic solvents can substantially increase activity.
It is also widely accepted that high concentrations of polymeric compounds promote rigidity in the structure of enzymes with which they are mixed. Hydrating these enzymes has the opposite effect where enzymes become less rigid and more flexible. Klibanov has referred to this phenomenon as ‘lubricating’ the enzyme. Hydration takes advantage of the ability of water to form hydrogen bonds with functional groups of a protein molecule, which may have been bound to each other before addition of water.
One way to change the concentration of inorganic salts and polymeric stabilizers is to reformulate the enzymes prior to use. Studies by Zaks and Klibanov have shown that hydration of powdered enzymes, prior to reaction with substrate, results in a loosening up of the enzyme structure and, at certain levels, the onset of catalytic activity. Unfortunately, most industrial enzyme users prefer liquid enzyme solutions to powdered enzymes. Shipping, transferring and hydrating dry enzymes is not the most efficient way to use enzymes especially when liquid enzymes can be stabilized with polymeric materials.
While hydration of enzymes may induce flexibility in their structures, in separate studies, Klibanov and Won found that increasing the water content in organic solvents to the water solubility limit, caused enzyme agglomeration resulting in glue-like fibers devoid of catalytic activity. Therefore, while hydration has the benefits listed above, there are also practical issues limiting the ability of enzyme users to hydrate enzymes in organic solvents.
Commercial enzyme preparations also contain a high concentration of enzymes, between 5 mg/mL and 25 mg/mL. These commercial enzyme preparations, have the benefit of reducing the number of shipments and the required storage capacity in facilities that use industrial enzymes.
Liquid enzyme formulations are often dosed at 3 places in an ethanol plant;                1) The slurry system, where initial hydrolysis takes place. In a typical 40 million gallon per year dry-mill ethanol plant, alpha-amylase is often added at between 500 mg/min and 1200 mg/min        2) The liquefaction system, where secondary hydrolysis takes place. In a typical 40 million gallon per year dry-mill ethanol plant, alpha-amylase is often added at between 1000 mg/min and 2000 mg/min        3) The fermentation system, where final hydrolysis and fermentation of the product takes place. In a typical 40 million gallon/year dry-mill ethanol plant, the enzyme dose is in the range of between 60 and 120 Gallons in a 500,000 Gallon fermenter.        
These dose ranges are adjusted accordingly for different plant capacities. For instance, 100 million gallon per year dry-mill ethanol plants require an alpha amylase dose in the range of 1250 mg/min and 3000 mg/min in the slurry system and between 2500 mg/min and 5000 mg/min in slurry and liquefaction respectively.
In addition, ethanol plants may produce ethanol from different types of feedstock. These feedstocks will vary in terms of the amount of ethanol produced per ton of feedstock. For example, dry mill ethanol plants typically produce between 2.5 and 2.9 Gallons per bushel of corn. The corn is milled and mixed with water in a ratio of between 28% and 38% solids. The theoretical ethanol yield for a ton of corn stover is 113 Gallons per dry ton. Currently, solids ratios for ethanol production from biomass sources such as corn stover are lower than solids ratios for ethanol production from corn and other grains and is typically between 8 and 20% solids.
However at high enzyme concentrations it is difficult to accurately dose low volumes of enzyme since, in the case of a 25 mg/mL protein, each milliliter contains 25 mg of protein, which may be more than one wants to dose over a particular time frame.
This problem is exacerbated by the polymeric stabilizers, which are characterized by high specific gravities. The combination of high specific gravity and high enzyme concentration makes it difficult to fine-tune dosing of industrial enzymes to bioreactors. Specialized pumps, capable of pumping high specific gravity liquids at low flow rates are expensive and there are limits to their accuracy.
Saville (U.S. published patent application No. 20040259219) showed that the activity of a group 3 hydrolase could be increased by diluting said group 3 hydrolase in water or an aqueous buffer and treating said hydrolase with activated carbon. Saville showed specifically that the activity increase was due to a reaction between the enzyme and the activated carbon. Saville's dilution step also reduces the concentration of salts and other preservatives, however, diluting with water or an aqueous buffer reduces the concentration of polymeric compounds to the point where ester-based or lactone-based polymers quickly form in the enzyme solution. Bacteria can grow on these polymers causing problems in bioreactors. These polymers coat the instrumentation and pipes, reducing flow and causing instrumentation to malfunction. In addition, increasing enzyme activity is not always desirable, for example in simultaneous saccharification and fermentation systems, an overly active glucoamylase enzyme will produce glucose at a rate that is detrimental to conversion of glucose to ethanol. These problems are exacerbated when the enzymes are diluted and treated with activated carbon in a central location, then delivered to enzyme users. Because Saville's purified enzymes are only stable for a short period of time, and Saville does not describe a way to purify enzymes on site and just-in-time, the invention by Saville is difficult to practice.
Laustsen (U.S. Pat. No. 6,582,606) teaches microfiltration of an enzyme solution using small amounts of activated carbon. While Laustsen does not teach increased activity, Laustsen does claim that microfiltration with activated carbon increases process capacity and reduces fouling.
However Laustsen's method teaches only a 1:1 and a 1:1.5 dilution of the enzyme solution prior to treatment with activated carbon. The specific gravity of an enzyme solution diluted 1:1 or 1:1.5 is still much higher than 1.0 g/mL, and poses problems for accurate dosing. In addition, Laustsen's process requires expensive microfiltration equipment, and which requires specialized expertise that may not be present in a carbohydrate processing operation. Finally, Laustsen's process specifies microfiltration of solids from liquid formulations. There are no solids in the commercial enzyme that is delivered to industrial users of Group 3 hydrolases in liquid form, therefore one skilled in the art would find little use in repeating the microfiltration of liquid enzymes as per Laustsen's invention.
Lab-scale assay demonstrates a significant decrease in enzyme activity for reformulated vs. non-reformulated enzymes, with reformulated enzymes being those having the concentration of enzyme and stabilizers reduced by dilution with water or aqueous buffer solutions. Since full scale production runs cost upwards of $150,000, those skilled in the art would avoid testing such reformulated enzymes on full scale production runs.
In light of the prior findings on loss of catalytic activity from over-hydration of enzymes in an organic solvent, a person skilled in the art would not reformulate an enzyme prior to delivery to a bioreactor, especially since enzyme users can cost-effectively add the enzyme and the polymeric materials to a bioreactor with no adverse effect. In addition, in light of reduced stability, increased bacterial growth and polymer formation, a person skilled in the art would hesitate to dilute an enzyme solution with aqueous buffer prior to adding to a bioreactor. Further, commercial enzyme solutions are free of solids, therefore one skilled in the art would not practice the mixing of commercial enzyme solutions and activated carbon prior to microfiltration through a membrane. Commercial enzyme preparations are already filtered through such membranes before they are received by the enzyme user; there would be substantial expense with little associated benefit.
There is a need to reformulate industrial enzymes to reduce the concentration of salts and other preservatives, decrease the specific gravity of the commercial enzyme preparation for accurate dosing, maintain stability of the solution for a timeframe long enough to deliver the reformulated solution to a bioreactor, prevent polymer growth and provide an enzyme solution with a desirable level of activity for the intended use. An effective reformulation method will also allow enzyme producers to provide higher strength commercial enzyme formulations to further reduce shipping costs and storage capacity requirements, both at the enzyme supplier facility and at the enzyme user facility. Users of commercial enzyme preparations can adapt to these higher strength enzyme formulations without acquiring new, more expensive and possibly less accurate pumps.